Throughout this application, various publications are referenced in parenthesis by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells. They hold promise for treatment of neurological, psychiatric, and endocrine disorders (46). Often the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals.
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant neuropeptide to be identified in mammalian brain. NPY is an important regulator in both the central and peripheral nervous systems (47) and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and have contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen (48), intestinal membranes, brain (49), aortic smooth muscle (50), kidney, testis, and placenta (2). In addition, binding sites have been reported in a number of rat and human cell lines (e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2), CHP-234, and SMS-MSN) (51,5).
Neuropeptide Y (NPY) receptor pharmacology is currently defined by structure activity relationships within the pancreatic polypeptide family (1, 2). This family includes NPY, which is synthesized primarily in neurons; peptide YY (PYY), which is synthesized primarily by endocrine cells in the gut; and pancreatic polypeptide (PP), which is synthesized primarily by endocrine cells in the pancreas. These 36 amino acid peptides have a compact helical structure involving a xe2x80x9cPP-foldxe2x80x9d in the middle of the peptide. Specific features include a polyproline helix in residues 1 through 8, a xcex2-turn in residues 9 through 14, an xcex1-helix in residues 15 through 30, an outward-projecting C-terminus in residues 30 through 36, and a carboxyl terminal amide which appears to be critical for biological activity (3). The peptides have been used to define at least four receptor subtypes known as Y1, Y2, Y3, and PP. Y1 receptor recognition by NPY involves both N- and C-terminal regions of the peptide; exchange of Gln34 with Pro34 is fairly well tolerated (3, 4, 5). Y2 receptor recognition by NPY depends primarily upon the four C-terminal residues of the peptide (Arg33- Gln34-Arg35- Tyr36-NH2) preceded by an amphipathic xcex1-helix (3, 6, 7); exchange of Gln34 with Pro34 is not well tolerated (4, 5). Y3 receptor recognition is characterized by a strong preference for NPY over PYY (8). Exchange of Gln34 in NPY with Pro34 is reasonably well tolerated by the Y3 receptor but PP, which also contains Pro34, does not bind well (8). The PP receptor is reported to bind tightly to PP, less so to [Leu31,Pro34]NPY, and even less so to NPY (3, 9). The only NPY receptor which has been cloned to date is the Y1 receptor gene, from mouse (12), rat (52), and human (10). One of the key pharmacological features which distinguish Y1 and Y2 is the fact that the Y1 receptor (and not the Y2 receptor) responds to an analog of NPY modified at residues 31 and 34 ([Leu31,Pro34]NPY), whereas the Y2 receptor (and not the Y1 receptor) has high affinity for the NPY peptide carboxyl-terminal fragment NPY-(13-36) (53,4).
Receptor genes for the other two structurally related peptides, peptide YY (PYY) and pancreatic polypeptide (PP), also have not been cloned. Peptide YY occurs mainly in endocrine cells in the lower gastrointestinal tract (54). Receptors for PYY were first described in the rat small intestine (55). This receptor has been defined as PYY-preferring because it displays a 5-10 fold higher affinity for PYY than for NPY (55,56). Recently, a cell line, PKSV-PCT, derived from the proximal tubules of kidneys, has been described to express receptors for PYY (57).
In the last few years only the rat and human Y1 cDNAs have been cloned (10, 11). This success was based on identifying the randomly cloned FC5 xe2x80x9corphan receptorxe2x80x9d (12). We now report the isolation by expression cloning of a human hippocampal Y2 cDNA clone and two rat Y2 clones and their pharmacological characterization.
This invention provides an isolated nucleic acid molecule encoding a Y2 receptor.
This invention also provides an isolated protein which is a Y2 receptor.
This invention provides a vector comprising nucleic acid encoding a Y2 receptor.
This invention also provides vectors such as plasmids comprising nucleic acid encoding a Y2 receptor, adapted for expression in a bacterial cell, a yeast cell, an insect cell or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the nucleic acid in the bacterial, yeast, insect or mammalian cells operatively linked to the nucleic acid encoding the Y2 receptor as to permit expression thereof.
This invention provides a cell transfected with and expressing nucleic acid encoding a Y2 receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a Y2 receptor.
This invention provide an antisense oligonucleotide having a sequence capable of specifically hybridizing with any sequences of an mRNA molecule which encodes a Y2 receptor so as to prevent translation of the mRNA molecule.
This invention provides an antibody directed to a Y2 receptor.
This invention provides a transgenic nonhuman mammal expressing nucleic acid encoding a Y2 receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a Y2 receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a Y2 receptor and which hybridizes to mRNA encoding a Y2 receptor thereby reducing its translation.
This invention further provides a transgenic nonhuman mammal comprising a homologous recombination knockout of the native Y2 receptor.
This invention provides a method for determining whether a ligand can bind specifically to a Y2 receptor which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 receptor with the ligand under conditions permitting binding of ligands to such receptor, and detecting the presence of any such ligand bound to the Y2 receptor, thereby determining whether the ligand binds specifically to a Y2 receptor.
This invention also provides a method for determining whether a ligand is a Y2 receptor agonist which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 receptor with the ligand under conditions permitting the activation of a functional Y2 receptor response from the cell, and detecting, by means of a bioassay, such as a second messenger assay, an increase in Y2 receptor activity, thereby determining whether the ligand is a Y2 receptor agonist.
This invention further provides a method for determining whether a ligand is a Y2 receptor antagonist which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 encoding with the ligand in the presence of a known Y2 receptor agonist, such as NPY, under conditions permitting the activation of a functional Y2 receptor response, and detecting, by means of a bioassay, such as a second messenger assay, a decrease in Y2 receptor activity, thereby determining whether the ligand is a Y2 receptor antagonist.
This invention further provides a method of screening drugs to identify drugs which specifically bind to a Y2 receptor on the surface of a cell which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 receptor with a plurality of drugs under conditions permitting binding of drugs to the Y2 receptor, and determining those drugs which bind to the Y2 receptor, thereby identifying drugs which specifically bind to a Y2 receptor.
This invention also provides a method of screening drugs to identify drugs which act as agonists of a Y2 receptor on the surface of a cell which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 receptor with a plurality of drugs under conditions permitting the activation of a functional Y2 receptor response, and determining those drugs which activate the Y2 receptor, using a bioassay, such as a second messenger assay, thereby identifying drugs which act as Y2 receptor agonists.
This invention also provides a method of screening drugs to identify drugs which act as antagonists of a Y2 receptor on the surface of a cell which comprises contacting a cell transfected with and expressing nucleic acid encoding the Y2 receptor with a plurality of drugs in the presence of a known Y2 receptor agonist, such as NPY, under conditions permitting the activation of a functional Y2 receptor response, and determining those drugs which inhibit the activation of the Y2 receptor, using a bioassay, such as a second messenger assay, thereby identifying drugs which act as Y2 receptor antagonists.
This invention also provides a method of detecting expression of a Y2 receptor by a cell by detecting the presence of mRNA coding for the Y2 receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding the Y2 receptor under hybridizing conditions, and detecting the presence of mRNA hybridized to the probe, thereby detecting the expression of a Y2 receptor by the cell.
This invention provides a method of determining the physiological effects of expressing varying levels of Y2 receptors which comprises producing a transgenic nonhuman mammal expressing nucleic acid encoding a Y2 receptor whose levels of Y2 receptor expression are varied by use of an inducible promoter which regulates Y2 receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of Y2 receptors which comprises producing a panel of transgenic nonhuman animals each expressing nucleic acid encoding a Y2 receptor expressing nucleic acid and expressing a different amount of Y2 receptor.
This invention provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific Y2 receptor allele which comprises: a. obtaining nucleic acid of subjects suffering from the disorder; b. performing a restriction digest of the nucleic acid with a panel of restriction enzymes; c. electrophoretically separating the resulting nucleic acid fragments on a sizing gel; d. contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to nucleic acid encoding a Y2 receptor and labeled with a detectable marker; e. detecting labeled bands which have hybridized to the nucleic acid encoding a Y2 receptor labelled with a detectable marker to create a unique band pattern specific to the nucleic acid of subjects suffering from the disorder; f. preparing nucleic acid obtained for diagnosis by steps a-e; and g. comparing the unique band pattern specific to the nucleic acid of subjects suffering from the disorder from step e and the nucleic acid obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
The invention provides a method of preparing an isolated, purified Y2 receptor which comprises constructing a vector adapted for expression in a cell which comprises the regulatory elements necessary for the expression of nucleic acid in the cell operatively linked to the nucleic acid encoding a Y2 receptor as to permit expression thereof, wherein the cell is selected from the group consisting of bacterial cells, yeast cells, insect cells and mammalian cells; inserting the vector of the previous step in a suitable host cell; incubating the cells under conditions allowing the expression of a Y2 receptor; recovering the receptor so produced and purifying the receptor so recovered.